Fig. 6

Adapting the GPI anchor to single-chain antibodies. a Vero cells were transfected with vehicle control or 1 μg of either RSV or Flu aVHH mRNA. After overnight incubation, cells were infected for 24 h before being fixed and stained for RSV N (red), panRSV (blue), and VHH (green). Scale bar represents 10 μm. b Quantification of mean volume of RSV N signal per cell from microscopy images in part (a). Error bars represent 95% confidence intervals. Asterisks indicate p < 0.0001 (Mann–Whitney U test). c Cells were transfected with vehicle control or 1 μg of RSV aVHH mRNA and infected with RSV at MOI of 0.1 before being analyzed by plaque assay. Error bars represent standard deviation. Asterisks indicate p < 0.01 (Mann–Whitney U test). d Cells were transfected with vehicle control or 1 μg of either aPali or RSV aVHH mRNA. Cells were infected with RSV for 24 h before RNA was isolated. Mean RSV F copy number was determined by qRT-PCR and normalized to the mock control group. Asterisk indicates p < 0.05 (Kruskal–Wallis test, Dunn’s multiple comparisons test compared to mock). e Mice lungs were transfected with saline or 100 μg of either naked RSV aVHH, aPali HC only, or Flu aVHH mRNA. aPali HC, and Flu aVHH controls, along with a RSV only group, were performed in a separate experiment – these data are repeated from Fig. 5a, b. Uninfected mice were sacrificed on days 1 and 5 post transfection. 1 day after transfection, mice were infected intranasally with RSV L19. Lungs were cryosectioned and stained for VHH (white) and panRSV (green). Scale bar represents 50 μm. f Infected mice lung homogenate supernatants from part (e) were analyzed by plaque assay. Error bars represent standard deviation. Asterisk indicates p < 0.05 (t-test, one-tailed). Hash indicates p = 0.07. g Infected mice lung homogenates from part (e) were analyzed by qRT-PCR for mean RSV F copy number. Data was normalized by RSV only control. Error bars represent standard deviation. Asterisk indicates p < 0.05 when compared to RSV (t-test, two-tailed)